Microtomographic, histomorphometric, and molecular features show a normal alveolar bone healing process in iNOS-deficient mice along a compensatory upregulation of eNOS and nNOS isoforms

Abstract Inducible nitric oxide synthase (iNOS) is one of the enzymes responsible for the synthesis of nitric oxide (NO), which is an important signaling molecule with effects on blood vessels, leukocytes, and bone cells. However, the role of iNOS in alveolar bone healing remains unclear. This study investigated the role of iNOS in alveolar bone healing after tooth extraction in mice. Methodology C57Bl/6 wild type (WT) and iNOS genetically deficient (iNOS-KO) mice were subjected to upper incision tooth extraction, and alveolar bone healing was evaluated by micro-computed tomography (μCT) and histological/histomorphometric, birefringence, and molecular methods. Results The expression of iNOS had very low control conditions, whereas a significant increase is observed in healing sites of WT mice, where iNOS mRNA levels peak at 7d time point, followed by a relative decrease at 14d and 21d. Regarding bone healing, both WT and iNOS-KO groups showed the usual phases characterized by the presence of clots, granulation tissue development along the inflammatory cell infiltration, angiogenesis, proliferation of fibroblasts and extracellular matrix synthesis, bone neoformation, and remodeling. The overall micro-computed tomography and histomorphometric and birefringence analyses showed similar bone healing readouts when WT and iNOS-KO strains are compared. Likewise, Real-Time PCR array analysis shows an overall similar gene expression pattern (including bone formation, bone resorption, and inflammatory and immunological markers) in healing sites of WT and iNOS-KO mice. Moreover, molecular analysis shows that nNOS and eNOS were significantly upregulated in the iNOS-KO group, suggesting that other NOS isoforms could compensate the absence of iNOS. Conclusion The absence of iNOS does not result in a significant modulation of bone healing readouts in iNOS-KO mice. The upregulation of nNOS and eNOS may compensate iNOS absence, explaining the similar bone healing outcome in WT and iNOS-KO strains.


Introduction
Bone repair occurs throughout coordinated and interconnected stages, starting with a temporary inflammatory response, which constructively influences the following events. 1 A low magnitude and transient production of inflammatory mediators in bone fracture sites supports osteogenic cellular differentiation and bone formation required for a positive healing outcome, being this concept supported by the causeand-effect association between anti-inflammatory drugs and delayed bone healing. 2 In this scenario, macrophages contribute to repairing and remodeling initially exerting pro-inflammatory effects associated with the M1 phenotype, followed by a phenotypic switch towards the dominance of the M2 type, which is anti-inflammatory and proreparative. [3][4][5] Within the multiple and functional M1 markers, inducible nitric oxide synthase (iNOS, also called NOS2) stands out as a prototypic M1 product.
iNOS comprises an inducible nitric oxide synthase isoform expressed in response to different stimuli usually described as the main factor responsible for triggering nitric oxide (NO) production in inflammatory environments. 6 Nitric oxide (NO) is a small and highly diffusible reactive molecule, with many functions, including its role as a second messenger on several inflammatory events. 7,8 Notably, although the production of NO can catalyze other nitric oxide synthases (NOS) isoforms, namely eNOS (endothelial NOS or NOS1) and nNOS (neuronal NOS or NOS3), 7,8 iNOS is the main NO inducer along inflammatory immune responses. iNOS is induced in many cell types in response to several inflammatory cytokines (such as TNF and IL1b), hypoxia, and oxidative stress. 9 These potential iNOS inducers are present in bone injury/healing sites, 7 reinforcing the involvement of iNOS as part of the inflammatory process that precedes bone healing.
Notably, iNOS/NO axis influence in bone healing can extend its inflammatory properties. NO is described as a potent regulator of bone cells, since it can exert both anabolic and anti-resorptive effects in the bone, with both pre-clinical and clinical data supporting the role of NO in bone health. 10,11 NO can inhibit the recruitment, proliferation, differentiation, activation, and/or survival of osteoclasts and their precursors in vitro. 12 Accordingly, iNOS deficiency can accelerate osteoclast formation and bone resorption, decrease normal bone mass, and exacerbate bone destruction in arthritis models. 12,13 Also, in vitro and in vivo evidences show NO in osteoblast differentiation and proliferation during bone development and healing. 11 Thus, considering the involvement of iNOS in inflammatory responses and its modulatory effects on bone cells, it is reasonable to hypothesize iNOS involvement in alveolar bone healing process. Indeed, iNOS is described to be expressed during mandibular bone healing in rabbits, and deletion of iNOS gene impairs mouse fracture endochondral healing. 14 However, despite the many in vitro studies, the few in vivo studies focused on the role of iNOS in bone repair remain to be complemented to properly address the real functions of iNOS in this process. In this context, this study investigated the role of iNOS intramembranous alveolar bone healing outcome, by the iNOS-KO and C57Bl/6-WT mice strain comparative analysis using micro-computed tomography (μCT) and histological, histomorphometric, immunological, and molecular methods. The animals were anesthetized by intramuscular administration of ketamine chloride (Dopalen, Agribrans Brasil LTDA) and of xylazine chloride (Anasedan, Agribrands Brasil LTDA) and the right upper incisor was extracted. At the end of the experimental periods (zero hours and seven, 14, and 21 days post extraction), the animals were euthanized with an excessive dose of anesthetic, and the maxillae were collected. Five maxillae were subjected to microcomputed tomography (μCT) and histological analyses;

Methodology
and four samples containing only the region of the alveolus were subjected to the Real-Time PCR array analysis (the molecular analysis also comprised an additional independent group of WT iNOS-KO mice, not subjected to any surgical procedure, used to perform data normalization). Due to limited availability of knockout mice, the μCT and picrosirius analyses were only performed at the 7d and 14d time points. Samples for the μCT and histological analyses were fixed in PBS-buffered formalin (10%) solution (pH 7.2) for 48 hours at room temperature, then washed overnight in running water and maintained temporarily in alcohol fixative (70% hydrous ethanol) until the conclusion of the μCT analysis, decalcified in 4.13% EDTA (pH 7.2) and subjected to histological processing. Samples for molecular analysis were stored in RNAlater (Ambion, Austin, TX) solutions.

Micro-computed tomography (μCT) evaluation
The maxillae samples were scanned as previously

Real-Time PCR array reactions
Real-Time PCR array reactions were performed as previously described. 4 The extraction of total RNA from the remaining alveolus was performed with the RNeasy

Matrix birefringence analyses
At 7d, the presence of granulation tissue was characterized by many newly formed small blood vessels, an intense inflammatory cell infiltrate, and immature connective tissue with many fibroblasts and immature collagen fiber bundles ( Figure 4A). At this time, fiber emitting birefringence in green tones (immature fibers) ( Figure 4A) was found in abundance. At 14d, the maturation of connective tissue was evidenced by the presence of mature collagen fiber bundles ( Figure 4B), when about three quarters of the fibers presented the red color spectrum, representative of a mature matrix ( Figure 4B). Comparing the percentage of fibers between different strains, the iNOS-KO mice had a higher amount of immature (green) fibers at 14d compared to the WT mice (p<0.05) ( Figure 4B). Similarly, yellow fibers were significantly more abundant in iNOS-KO mice than WT mice during all experimental periods (p<0.05) ( Figure   4B), whereas mature fibers (red) were significantly more abundant in WT mice (p<0.05) ( Figure 4B).  Samples from C57BL/6 wild-type (WT) and iNOS-KO mice were subjected to Real-Time PCR array analysis at zero, seven, 14, and 21 days post tooth extraction to evaluate the kinetics of gene expression of the bone healing process. Molecular analysis (Real-Time PCR array) using a heat map to quantify the expression of the ECM and bone formation markers, osteoclast markers, inflammatory cytokines, M1 and M2 markers, and iNOS isoforms eNOS and nNOS were evaluated. Results are expressed as fold change relative to a normalizing control (C, i.e., normal alveolar bone) sample and normalized by three constitutive genes (GAPDH, ACTB, Hprt1); * represents the differences (p<0.05) between iNOS and WT group at each time point Initially, our results showed that iNOS expression is significantly upregulated in alveolar bone healing sites.

Kinetics analysis of gene expression patterns in the bone healing process
Accordingly, previous studies describe the upregulation of iNOS in similar contexts, such as in bone fracture sites 13 . When iNOS expression profile is compared with the migration pattern of M1 cells in healing sites, 1,4,16,17 we can observe a similar kinetics pattern between iNOS levels and CD80+ cells, reinforcing the possible iNOS/ M1 interconnection along the bone healing process.
Potential iNOS inducers, such as pro-inflammatory cytokines TNF, IL1B and IL6, and M1-markers, 1,4,[16][17][18] were expressed in bone healing sites with profiles resembling that observed in iNOS, also strengthening the putative involvement of iNOS as part of the inflammatory process that precedes bone healing.
However, the comparison of bone healing parameters in WT and iNOS-KO showed an overall similarity of healing kinetics and outcome in both strains. We observed no differences between the iNOS-KO and WT strains by the three-dimensional images from the μCT of maxillae, which had similar bone microarchitecture features upon quantitative evaluation of μCT reconstruction.
Notably, bone healing in both the iNOS-KO and WT strains was similar to that previously described in the WT C57Bl/6 strain. 1,4,16,17 Since only mineralized tissue features are subjected to the μCT analysis, And an additional hypothesis is that differential iNOS levels in such a distinct environment can influence the distinct outcomes, since the expression of iNOS in chronic inflammatory osteolytic lesions is significantly higher than in healing sites (data not shown).